Most HIV transmissions occur mucosally, including mother-to-child transmission (MTCT). As HIV clade C predominates worldwide, we initially focused on immunoprevention of HIV clade C MTCT. We identified a quadruple (4x) combination of human neutralizing monoclonal antibodies (nmAbs) that, although raised against clade B, was broadly reactive across clades, including clade C. To test the 4 nmAbs in vivo, we constructed several simian-human immunodeficiency viruses encoding env of primary R5 HIV clade C strains (termed SHIVenvC's). The 4x nmAb combination prevented infection in 3 out of 4 monkey infants, whereas all controls had high peak viremia after oral SHIVenvC challenge. While we provided compelling evidence for the protective role of nAbs, we and others were stunned by findings that some of these 4 nmAbs are auto-antibodies. We re-oriented our HIVRAD goals as there is a great need to find new targets for nmAb responses, especially against HIV clade C. This re-competition has three main goals: 1. To refine the SHIVenvC primate model for mucosal transmission by increasing the virulence of R5 SHIVenvC's, generating a fully heterologous SHIVenvC, and by comparing the molecular evolution of HIV clade C env in infected African mother/infant pairs and in rhesus monkeys infected with SHIV's harboring homologous HIV clade C env genes. 2. To harness the broadly reactive humoral immune responses of monkeys infected with SHIVenvC's whose sera neutralize divergent HIV clade C and B strains. Using B cells of such monkeys, we seek to isolate nmAbs, determine epitope specificities, and exploit random peptide phage-display libraries to identify mimotopes to the novel rhesus nmAb epitopes, both linear and conformational (via a novel computer program). We will also directly use phage display with polyclonal IgG with broadly reactive nAb responses. 3. To test the new concept of SEquential Editing and Focusing of Antibody Responses (SEEFAR). We postulate that the variable (V) loops in Env attract most of the nmAb responses, which predominantly target individual virus types. We seek to avoid boosting such narrow, type-specific nAb responses by developing Env immunogens deleted in most/all V loops, and/or sequentially editing out type-specific nAb responses by boosting with immunogens that do not share V-loop epitopes. The SEEFAR strategy will be tested in the context of a replication-competent adenovirus prime/recombinant protein boost. Some of the reagents and vaccine strategies proposed can be entered directly into future clinical trials, and insights gained from analyzing broadly reactive nmAbs will provide important new structural information for the subsequent design of improved immunogens. Lastly, fine-tuning the SHIVenvC primate model will allow testing candidate anti-HIV clade C AIDS vaccines in a relevant mucosal challenge system. PROJECT 1: Molecular Evolution of HIV Clade C Env in Two Species (Wood, C.) PROJECT 1 DESCRIPTION (provided by applicant): The development of an effective HIV-1 vaccine has been hampered by the rapid rate of mutation and genetic variation throughout the viral genome, especially the env gene. In this proposal, we seek to analyze the evolution of HIV clade C env genes in children that were perinatally infected. In parallel, we will analyze the evolution of env genes in rhesus monkeys infected with SHIV strains encoding the corresponding HIV clade C env genes. We hypothesize that the molecular evolution of env genes in the two different species will follow similar patterns. Together with Core A, we will generate new SHIV strains that encode env genes from HIV clade C-infected Zambian infants with rapid HIV disease progression. We hypothesize that Env is a determinant for pathogenicity, which will be mirrored in the corresponding SHIV strains in macaques. The overall objective of Project 1 is to determine whether infection of non-human primates by SHIV and the resultant molecular evolution of env therein, is a suitable model for studying HIV-1 evolution in humans. Our immediate goal is to longitudinally isolate samples from perinatally infected infants and to characterize the patterns and within-host diversification of clade C env genes. Using nucleotide sequence data from viral isolates obtained in parallel from infected humans and macaques, we will test several hypotheses regarding the relationships among genetic diversity, disease progression, and clinical phenotypes. This proposal will make use of an ongoing research project to study perinatal transmission of HIV and other viruses in mother/infant pairs (MIPs) from Zambia. The Specific Aims are: 1) To characterize changes in Env proteins of HIV-1 clade C isolated from infants at different time points in the disease course, and correlate these changes to disease progression. 2) To characterize Env proteins of SHIV clade C isolated longitudinally over the disease course in infected animals, and correlate these changes to disease progression in both the animals and the patients. 3) To characterize the biological properties of Env proteins of viruses obtained from the paired patients and macaques with time, and correlate these changes to disease progression. This study will provide important predictive information about the evolution of HIV clade C by correlating patterns of env evolution with pathogenesis and disease progression. This study will also lead to a better understanding of the host-virus interaction during the disease course and will provide useful information for designing vaccines.